1. Field of the Invention
The present invention relates to apparatus and methods for capture, separation, manipulating, measurement, and analysis of micro and nanoparticles and molecular targets.
2. Related Art
Biophysics has been revolutionized in the past decade. In particular, single molecule biophysics allows us to study many biological reactions in quantitative and physical standpoint by directly measuring physical properties of and mechanical interactions among biological molecules (biomolecules) such as DNA and proteins (Svoboda and Block 1994; Ludwig 1999; Mehta, Rief et al. 1999; Bustamante, Macosko et al. 2000). Such a breakthrough was made possible by a number of new methods for manipulating single biomolecules. New manipulation tools are making it possible to follow, in real time and at a single molecule level, the movements, forces, and strains that develop during the course of a reaction because they can exert external forces at appropriate levels to modify the conformation of biomolecules, and highly sensitive detectors can measure the forces and displacements with high spatial and temporal resolutions.
These innovative tools expand the horizon of research areas and convert biological problems that were previously intractable into answerable questions. These biological problems include protein folding (Carrion-Vazquez, Oberhauser et al. 1999), DNA elasticity (Smith, Finzi et al. 1992; Marko 1995), the protein-induced bending of DNA (Erie, Yang et al. 1994), the stress-induced catalysis of enzymes (Wuite, Smith et al. 2000), the behavior of molecular motors (Kishino and Yanagida 1988; Howard, Hudspeth et al. 1989; Ishijima, Doi et al. 1991; Svoboda, Schmidt et al. 1993; Strick, Croquette et al. 2000; Wuite, Smith et al. 2000; Smith, Tans et al. 2001; Stone, Bryant et al. 2003), the protein-protein interaction (Nakajima, Kunioka et al. 1997), and the protein-induced DNA condensation (Case, Chang et al. 2004). Hence, developing new experimental methods is as crucial as clarifying mechanisms of individual biological phenomena in advance of biology.
Among many ingenious methods for single molecule biophysics, laser tweezers and magnetic tweezers are the two most interesting ones. Different from scanning force microscopy (Rief, Gautel et al. 1997; Carrion-Vazquez, Oberhauser et al. 1999) and glass needle method (Ishijima, Doi et al. 1991; Cluzel, Lebrun et al. 1996), the two methods can apply physiologically relevant, low level of force to biomolecules in biological environment. Different from hydrodynamic manipulation (Perkins, Smith et al. 1995), they can control biomolecules in well-defined, fast, and sophisticated manner.
Although magnetic tweezers are superior to laser tweezers in applying torque and sub-pN level of force and are much simpler than laser tweezers, it has remained a rather complementary tool to laser tweezers due to low upper limit of force and geometrical restriction that results in slow position measurement of the vertical dimension Z through diffraction ring analysis (Gosse and Croquette 2002). Another basic design for the magnetic tweezers was reported by Strick, Allemand et al., The elasticity of a single supercoiled DNA molecule, Science; 1996 Mar. 29; 271(5257):1835-7. Since magnetic force pulls magnetic-bead-tagged molecules from the above in most magnetic tweezers apparatus, important information such as DNA extension should be extracted from the Z position measurement (Strick, Allemand et al. 1996).
In order to overcome such obstacles, numerous modifications have been adopted. To increase the force maximum, a bigger bead with higher magnet content was used; a large drag on such beads makes their response slower, which limits the time resolution in the experiment. As an alternative approach, a tiny magnet piece was placed in proximity of a magnetic-bead-tagged molecule to obtain ˜200 pN with 2.8 μm magnetic bead {Yan and Marko 2004}. In this method, the extension of interest appears on the view plane similar to laser tweezers and is therefore easier to measure. In spite of these advantages, torque cannot be applied, force calibration via the calibration of pipette stiffness is required each time of experiment, and the system is more sensitive to environmental noise because the work has been demonstrated in open cell geometry.
One way of alleviating the aforementioned geometrical restriction is to change the geometry so that the position measurement on the view plane (X and Y) yields sufficient information to understand the conformation of biomolecules (Leuba, Karymov et al. 2003; Zlatanova and Leuba 2003). For the goal, a horizontal force component can be introduced from buffer flow or asymmetrically positioned magnets. The horizontal force component will tilt magnetic-bead-tagged molecules, which alleviates the need to analyze the vertical dimension and therefore speeds up the analysis. However, this method relies on additional measurements or assumptions.
As a new method, electromagnets have been employed in magnetic tweezers for better control of magnetic field and the position of magnetic particles (Haber 2000; Gosse and Croquette 2002). With electromagnets, even purely horizontal force can be generated by sophisticated feedback operation of 6 electromagnets and canceling gravitational force (Gosse and Croquette 2002). Although it permits fast and complex operations for positioning a particle precisely in 3D, the force is weak and electromagnets will require extensive cooling for higher force. All these modifications have overcome some of the obstacles but produced new ones.
A related hybrid magnet structure was previously developed in Lawrence Berkeley National Laboratory and Joint Genome Institute (JGI: Department of Energy) for use in biotechnology applications and is described in now issued U.S. Pat. No. 6,954,128 and continuation-in-part U.S. patent application Ser. No. 11/248,934, filed on Oct. 11, 2005, and is hereby incorporated in its entirety.
Herein are described hybrid magnetic tweezers and its use as a more powerful and versatile tool. A new analysis scheme utilizing Hilbert transformation makes it fast to determine the Z position in spite of the same geometrical restriction.